Project Summary/Abstract Eukaryotic genomes must simultaneously be packaged to fit into the cell nucleus, but also provide access at specific loci to allow for fundamental biological processes including gene transcription and genome replication. To accomplish these opposing requirements for packaging and access, eukaryotic genomes are regulated at many levels and length scales, from the nucleosome to the higher-order, three-dimensional interactions among chromosomes. My laboratory is investigating two different levels of regulation along this broad but interconnected spectrum: First, we are testing for the first time the extent of regulation of genome function at the level of nucleosome symmetry. Nucleosomes contain two copies of each core histone, held together by a naturally symmetric, homodimeric histone H3-H3 interface. This symmetry has complicated efforts to determine the regulatory potential of this architecture. In other words, is it important whether one or both tails receives a post- translational modification? Answering this question requires the ability to specifically impair modification on a single tail per nucleosome. Through molecular design and in vivo selection, we have generated obligately heterodimeric H3s, providing a unique tool for discovery of the degree to which histone modification symmetry plays a regulatory role in gene expression and other chromosomal functions in living cells. Having validated an asymmetric H3 pair, we are extending these studies to two additional H3 isoforms. First, we recently generated an asymmetric centromeric H3 (Cse4/CENP-A) pair in budding yeast. Using these, we will address long-standing controversies regarding centromeric nucleosome stoichiometry. Second, we are using an asymmetric replication-independent histone H3.3 pair to probe two histone modifications with key roles in chromatin structure and gene regulation. Histone H3.3 is required for repression of endogenous retrovirus transcription and early differentiation in mouse embryonic stem cells, so we plan to investigate the stoichiometry of regulatory relationships for repressive chromatin mechanisms that are absent in yeast, most notably involving H3K9me3 (characteristic of constitutive heterochromatin) and H3K27me3 (characteristic of facultative heterochromatin that is developmentally regulated). Because dominant H3.3 mutations are implicated in several types of cancer, these studies also provide a novel tool for exploration of how these alterations affect epigenomes in living cells. Second, we are exploring interconnections between the three-dimensional organization of the human genome, cell cycle progression, and protection from genotoxic stress. Our experiments have led us to focus on the clinically important proliferation marker protein Ki-67. Ki-67 is required for normal three- dimensional organization of heterochromatic loci around the nucleoli, protects cells from genotoxic stress, and is essential for forming a proteinaceous layer on mitotic chromosomes. It is not understood how Ki-67 contributes to these processes, or how these functions may be interrelated. We recently discovered that in human cells with intact G1/S cell cycle checkpoints, acute depletion of Ki-67 induces cell cycle inhibitor p21, reduces G1/S-regulated RNA levels, and delays S phase entry. These cell cycle phenotypes are accompanied by reduced maintenance of heterochromatin marks (e.g. H3K27me3) on the inactive X (Xi) chromosome in female checkpoint-proficient cells. Notably, all of these phenotypes are absent in cells lacking G1/S checkpoints. In other words, Ki-67 links cell cycle progression and chromosome maintenance in primary cells, and checkpoint-defective tumor cells evade these mechanisms. To begin molecular exploration of these novel functions, we will therefore test for molecular hallmarks of DNA damage upon Ki-67 depletion in checkpoint-proficient cells. We will also map which Ki-67 protein domains are required for its novel activities, and determine if they are separable from previously described roles in mitotic chromosome structure and interphase heterochromatin localization. In this manner, we will be poised to pursue relevant partner proteins on our path to new insights into the coordination of human chromosome structure and function.